Chemical synthesis of methoxy nucleosides

ABSTRACT

Process for chemical synthesis of methoxy nucleosides.

[0001] This patent application is a continuation of Beigelman et al.,U.S. Ser. No. 09/586,345, filed Jun. 2, 2000, which is a continuationBeigelman et al., U.S. Ser. No. 09/361,484, filed Jul. 26, 1999, andthis application is also a continuation-in-part of Beigelman et al.,U.S. Ser. No. 08/600,429 filed Feb. 13,1996. These applications arehereby incorporated by reference herein in their entirety including thedrawings.

BACKGROUND OF THE INVENTION

[0002] This invention relates to the chemical synthesis of 2′-O-methyl,3′-O-methyl and 5′-O-methyl nucleosides.

[0003] The following is a brief description of synthesis of methoxynucleosides. This summary is not meant to be complete but is providedonly for understanding of the invention that follows. This summary isnot an admission that all of the work described below is prior art tothe claimed invention.

[0004] Sugar modifications, such as 2′-O-methyl, have been discovered ina variety of naturally occurring RNA (e.g., tRNA, mRNA, rRNA; reviewedby Hall, 1971 The Modified Nucleosides in Nucleic Acids, ColumbiaUniversity Press, New York; Limbach et a!., 1994 Nucleic Acids Res. 22,2183). In an attempt to understand the biological significance,structural and thermodynamic properties, and nuclease resistance ofthese sugar modifications in nucleic acids, several investigators havechemically synthesized nucleosides, nucleotides and phosphoramiditescontaining various sugar modifications and incorporated them intooligonucleotides. There are several reports in the literature describingthe synthesis of 2′-O-methyl nucleosides, 2-O-methyl nucleotides,2′-O-methyl phosphoramidites and oligonucleotides containing 2′-O-methylsubstitutions (Broom and Robins, 1965 J. Am. Chem. Soc. 87, 1145; Martinet a!., 1968 Biochemistry, 7, 1406; Robins et a!., 1974 J. Org. Chem.39, 1891; Inoue eta!., 1987 NucleicAcids Res. 15, 6131; Cotten eta!.,1991 Nucleic Acids Res. 19, 2629; Andrews et al., 1994 J. HeterocyclicChem. 31, 765; Beigelman eta!., 1995 Nuc!eosides & Nucleotides 14, 421;Sproat et a!., 1990 Nucleic Acids Res. 18, 41).

[0005] Broom and Robins, 1965 J. Am. Chem. Soc. 87,1145 and Martin etal., 1968 Biochemistry, 7, 1406, describe the synthesis of 2′-O-methylribonucleotides involving. mono-methylation of a 2′,3′-cis-diol systemof a ribonucleoside with diazomethane. This procedure gives rise to amixture of 2′- and 3′-O-methyl nucleosides in 20-40% combined yield. Thetwo isomers are then separated by ion-exchange chromatography.

[0006] Robins et al., 1974 J. Org. Chem. 39, 1891, describe thetreatment of a methanolic solution of uridine with diazomethane (inglyme) in the presence of stannous chloride dihydrate (in methanol) tosynthesize 2′-O-methyluridine (58% yield). This reaction also yielded asignificant fraction (28%) of 3′-O-methyluridine which is purified awayfrom the 2′-O-rnethyl form by chromatography.

[0007] Inoue, Japanese Patent Publication No. 61291595 and Inoue et a.,1987 Nucleic Acids Res. 15, 613, describe a process for the synthesis of2′-O-methyl ribonucleosides involving alkylation of3′,5′-O-(tetraisopropyldisiloxane-1, 3-diyl) (TIPDS)-ribonucleosideswith methyl iodide. Inoue et al., state that (page 6133, second mainparagraph):

[0008] “Treatment of 3′,5′-O-TIDPS-uridine (1) with benzoyl chloride . .. in N,N-dimethylacetamide in the presence of triethylamine . . .selectively gave the N³-benzoylated derivative (2) in 70.5% yield. Then,2 was treated with CH₃l . . . in benzene in the presence of Ag₂O . . .at 40° C. overnight to give the N³-benzoyl-2′-O-methyl derivative (3,84.5%). Debenzoylation of 3 with dil. NH₄OH followed by removal of TIPDSgroup with 0.5N HCl afforded 2′-O-methyluridine . . . in 84% yield.”

[0009] Srivastava and Roy, U.S. Pat. No. 5,214,135, describe thesynthesis of 2′-O-methyl nucleosides using an approach similar to Inoueet al., supra, except that the reaction with methyl iodide/silver oxidewas carried out at 25° C. for 24-46 hr with an 80-86% yield. Thisreaction, similar to the one described by Inoue et al., supra, also gaverise to the 3′-O-methyl isomer in 6-8% yield.

[0010] Parmentier et al., 1994 Tetrahedron 50, 5361, describe aconvergent synthesis of 2′-O-methyl uridine. This procedure uses amulti-step process involving—“a facile obtention of the 2′-O-methylsugar synthon using totally selective and efficient methylationconditions; . . . a stereoselective high-yield condensation with anuracil derivative, yielding the desired 5-form with a satisfactoryanomeric excess.” (page 5361, fifth paragraph).

[0011] Chanteloup and Thuong, 1994 Tetrahedron Letters 35, 877,-describesynthesis of 2′-O-alkyl ribonucleosides using trichloroacetimidate D-ribofuranosides as ribosyl donors. They state in the abstract on page877—

[0012] “Trichloroacetimidate-2-O-alkyl-3,5-0-TIPS-β-D-ribofuranosideglycosylates silylated nucleobases in a fast high-yielding andstereoselective reaction promoted by trimethylsilyltrifluoromethanesulfonate. This method has been applied to the synthesisof 2′-O-alkyl ribonucleosides further transformed to building blocksready for oligo(2′-O-alkyl)ribonucleotide construction.”

[0013] Beigelman et al., 1995 Nucleosides & Nucleotides 14, 421,describe three different approaches to the synthesis of 2′-O-methylnucleosides. They state that—

[0014] Method 1:

[0015] “Retrosynthetic analysis showed that 3-O-alkylated derivatives of1,2:5,6-di -O-isopropylidene(IP)-α-D-allofuranose (1) could betransformed to the related 2′-O-alkyl ribofuranosides by selectivedegradation of the C₁-C₂ bond with subsequent cyclization of thegenerated C2-formyl group to the C5—OH.” (Page 421, third paragraph)

[0016] Method 2:

[0017] “The 3—-O-TBDMS-derivatives of protected ribonucleosides arebyproducts obtained during the preparation of 2′-O-TBDMS derivatives—keybuilding blocks in oligoribonucleotide synthesis. At the same time,3′-O-TBDMS-isomers could be useful starting compounds in the preparationof 2′-O-methyl-3′-O-phosphoramidites. We explored this possibility oncytidine derivative 14. Reaction of 3′-O-TBDMS-5′-O-DMT-N⁴-i-Bu-cytidine (14) with Ag₂O—CH₃I using a modified method ofOhtsuka et al. (supra) yielded 3′-O-TBDMS-5′-O-DMT-N⁴-i-Bu-2′-O-methylcytidine (15) in 26% yield. The 2′-O-TBDMS isomer 16 was also obtained(22% yield) along with the starting 3′-O-isomer (18%). When2′-O-TBDMS-5′-O-DMT-N⁴-i-Bu-cytidine (16) was subjected to the samereaction conditions, the same mixture of products was obtained. Theseresults show that under the above reaction conditions migration of theTBDMS group accompanies the methylation reaction and methylation takesplace selectively at the 2′-OH position.” (Page 422, second fullparagraph)

[0018] Method 3:

[0019] “Among different methods of indirect introduction of a methylgroup, the use of 1-alkylthioalkyl intermediates seems to be the mostpromising. Although methods of synthesis of methylthiomethyl ethers ofnucleosides and carbohydrates are well developed, their transformationinto a methyl group sometimes requires additional steps. We wereinterested in the testing of more reactive methylthiophenyl ethers asprecursors for methyl ethers. We found that methylthiophenyl etherscould be smoothly introduced by treating appropriately protectednucleosides or carbohydrates with PhSMe/Bz₂O₂ in the presence of DMAP.Nucleoside 19 afforded methythiophenyl ether 20 in 65-70% yield, andα-ribofuranose 21 was transformed into β-furanose 22 in 60% yield.Different attempts to radically (Bu₃SnH, Bz₂O²) reduce the thiophenylgroup of furanose 22 were not successful, providing only startingmaterial. However, under the same conditions, nucleoside 20 afforded2′-O-Me derivative 24 in 70% yield.

[0020] Haga et al., 1972 Carbohydrate Res. 21, 440 describe a “facileroute” to the synthesis of 2- and 3-O-methyl-D-ribose from3-O-methyl-D-allose.

[0021] Nair et al., 1982, Synthesis 8, 670, describes modification ofnucleic acid bases via radical intermediates.

[0022] Leonard et al., 1992, Nucleosides & Nucleotides, 11, 1201,describe a method for the preparation of protected 2′-O-methylguanosine.This procedure is distinct from the one described in the instantinvention.

[0023] Wagner et al., 1991, Nucleic Acids Res., 19, 5965, describes amethod for alkylation of ribonucleosides.

[0024] The information disclosed in the references cited above aredistinct from the presently claimed invention since they do not discloseand/or contemplate the processes for the synthesis of the methoxynucleosides as claimed in the instant invention.

SUMMARY OF THE INVENTION

[0025] It has been postulated (Ueda, in Chemistry of Nucleosides andNucleotides ed. L. B Townsend, v.1 Plenum Press 1988 pp.1-95) thatprotonation of the N₃ atom of 2,2′-, 2,3′ or 2,5′-anhydro pyrimidinenucleosides facilitates anhydro ring opening by different nucleophilesproducing, in most cases, nucleoside analogs containing modifications inthe carbohydrate portion of the nucleoside. Complexation of the N₃ atomof the above-mentioned anhydro derivatives with Lewis acids [e.g.B(OMe)₃] would provide the same effect directly or in. the case ofmethanolysis, complexation of the MeOH with Lewis acids would acidifythe related proton leading to potential protonation of the N₃ atom ofthe above-mentioned anhydro derivatives. Applicant investigatedmethanolysis of 2,2′-, 2,3′ or 2,5′-anhydro pyrimidine nucleosides inthe presence of a Lewis acid, such as B(OMe)₃ and/or BF₃.MeOH. Thereaction involving a 2,2′-anhydro-1 (β-D-arabinofuranosyl) nucleoside,such as 2,2′-anhydro-1(β-D-arabinofuranosyl) uracil or 2,2′-anhydro-1(β-D-arabinofuranosyl) cytosine, with B(OMe)₃ and/or BF₃.MeOH, resultsin the production of 2′-O-methyl nucleosides with a yield of about90-100%.

[0026] By “Lewis Acid” is meant a substance that can accept an electronpair from a base. Examples of Lewis acids are, B(OCH₃)₃, BF₃, AICI₃, andSO₃.

[0027] In one aspect, the invention features a process for the synthesisof a 2′-methyl adenosine nucleoside, comprising the step of contacting asolution of N⁴-acetyl-5′,3′-di-O-acetyl-2′-O-methyl cytidine with aLewis acid under conditions suitable for the formation of saidnucleoside.

[0028] In another aspect, the invention features a process for thesynthesis of 2′-O-methyl guanosine nucleoside, comprising the steps of:a) methylating 2-amino-6-chloropurine riboside by contacting said2-amino-6-chloropurine riboside with sodium hydride, dimethylformamideand methyl iodide under conditions suitable for the formation of2′-O-methyl-2-amino-6-chloropurine riboside; b) contacting said2′-O-methyl-2-amino-6-chloropurine riboside with 1,4-diazabicyclo(2.2.2)octane and water under conditions suitable for the formation of said2′-O-methyl guanosine nucleoside in a crude form; and c) purifying said2′-O-methyl guanosine nucleoside from said crude form.

[0029] In another aspect, the invention features a process for thesynthesis of 2′-O-methyl adenosine nucleoside, comprising the steps of:a) methylating 2-amino-6-chloropurine—riboside by contacting said2-amino-6-chloropurine riboside with sodium hydride, dimethylformamideand methyl iodide under conditions suitable for the formation of2′-O-methyl-2-amino-6-chloropurine riboside; b) contacting said2′-O-methyl-2-amino-6-chloropurine riboside with acetic anhydride,4-dimethylaminopyridine and triethylamine under conditions suitable forthe formation of 3′,5′-di-O-acetyl-2-O-methyl-6-chloro-2-aminopurineriboside; c) deaminating said3′,5′-di-O-acetyl-2′-O-methyl-6-chloro-2-aminopurine riboside withisoamyl nitrite and tetrahydrofuran to form3′,5′-di-O-acetyl-2′-O-methyl-6-chloropurine; d) aminating said3′,5′-di-O-acetyl-2′-O-methyl-6-chloropurine with ammonia to form2′-O-methyl adenosine nucleoside in a crude form; and e) purifying said2′-O-methyl adenosine nucleoside from said crude form.

[0030] In yet another aspect, the invention features a process for thesynthesis of 2′-O-methyl guanosine nucleoside, comprising the steps of:a) contacting 2,6-diaminopurine nucleoside with anhydrous pyridine andtetraisopropyl D-silyl chloride under conditions suitable for theformation of 2,6-diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-β-D-ribofuranosyl) purine; b)methylating said2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-β-D-ribofuranosyl) purine by contacting said 2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-β-D-ribofuranosyl) purine withanhydrous DMF and methyl iodide under conditions suitable for theformation of2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2′-O-methyl-β-D-ribofuranosyl) purine; c) acylating said 2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2-O-methyl-β-D-ribofuranosyl)purine by contacting said2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2-O-methyl-β-D-ribofuranosyl) purine with anhydrous pyridine and isobutyrylchloride under conditions suitable for the formation of2,6-Diamino-N²-isobutyryl-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2′-O-methyl-β-D-ribofuranosyl) purine; d) deaminating and desilylating said2,6-Diamino-N²-isobutyryl-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2-O-methyl-β-D-ribofuranosyl) purine under conditions suitable for the formation ofN²-isobutyryl-2′-O-methyl guanosine nucleoside in a crude form; e)purifying said N²-isobutyryl-2′-O-methyl guanosine nucleoside from saidcrude form; and f) deblocking said N²-isobutyryl-2′-O-methyl guanosinenucleoside under suitable conditions to form said 2′-O-methyl guanosinenucleoside.

[0031] In one aspect, the invention features a process for the synthesisof 2′-O -methyl guanosine nucleoside, comprising the steps of: a)contacting 2,6-diaminopurine nucleoside with anhydrous pyridine andtetraisopropyl D-silyl chloride (TIPSCI) under conditions suitable forthe formation of2,6-Diamino-9-(3′,5′-O-tetraisopropyldisiloxane-(1,3-diyl)-β-D-ribofuranosyl)purine; b) methylating said2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-β-D-ribofuranosyl) purine by contacting said 2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-β-D-ribofuranosyl) purine withanhydrous DMF and methyl iodide under conditions suitable for theformation of2,6-Diamino-9-(3′,5′-O-tetraisopropyldisiloxane-(1,3-diyl)-2′-O-methyl-β-D-ribofuranosyl) purine; c) acylating said 2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2-O-methyl-β-D-ribofuranosyl)purine by contacting said2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2-O-methyl-β-D-ribofuranosyl) purine with anhydrous pyridine andisopropylphenoxyacetyl chloride under conditions suitable for theformation of2,6-Diamino-N²-isopropylphenoxyacetyl-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2-O-methyl-β-D-ribofuranosyl) purine; d) deaminating anddesilylating said 2,6-Diamino-N²-isopropylphenoxyacetyl-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2-O-methyl-β-D-ribofuranosyl)purine under conditions suitable for the formation ofN²-isopropylphenoxyacetyl-2′-O -methyl guanosine nucleoside in a crudeform; e) purifying said N²-isopropylphenoxyacetyl-2′-O-methyl guanosinenucleoside from said crude form; and f) deblocking saidN²-isopropylphenoxyacetyl-2′-O-methyl guanosine nucleoside undersuitable conditions to form said 2′-O-methyl guanosine nucleoside.

[0032] In one aspect, the invention features a process for the synthesisof 2′-O-methyl guanosine nucleoside, comprising the steps of: a)contacting guanosine with N,N-dimethylformamide dibenzyl acetal underconditions. suitable for the formation of N1-benzyl guanosine; b)methylating said N1-benzyl guanosine by contacting said N1-benzylguanosine with silver acetylacetonate, trimethylsulphonium hydroxide anddimethylformamide under conditions suitable for the formation ofN1-benzyl-2′-O-methyl guanosine in a crude form; c) purifying saidN1-benzyl-2′-O-methyl guanosine from said crude form, d) removing theN1-benzyl protection from said N1-benzyl-2′-O-methyl guanosine bycontacting said N1-benzyl-2′-O-methyl guanosine with sodium naphthaleneunder conditions suitable for the formation of 2′-O-methyl guanosinenucleoside in a crude form; and e) purifying said 2′-O-methyl guanosinefrom said crude form.

[0033] In yet another aspect, the invention features a process for thesynthesis of 2′-O-methyl adenosine nucleoside, comprising the steps of:a) methylating adenosine by contacting said adenosine withdimethylformamide, silver acetylacetonate and trimethylsulphoniumhydroxide under conditions suitable for the formation of 2′-O-methyladenosine in a crude form; and b) purifying said 2′-O-methyl adenosinefrom said crude form.

[0034] In one aspect, the invention also features a process for thesynthesis of 2′-O-methyl guanosine nucleoside, comprising the steps of:a) contacting guanosine with N,N-dimethylformamide dibenzyl acetal underconditions suitable for the formation of N1-benzyl guanosine; b)methylating said N1-benzyl guanosine by contacting said N1-benzylguanosine with magnesium acetylacetonate, trimethylsulphonium hydroxideand dimethylformamide under conditions suitable for the formation ofN1-benzyl-2′-O-methyl guanosine in a crude form; c) purifying saidN1-benzyl-2′-O-methyl guanosine from said crude form; d) removing theN1-benzyl protection from said N1-benzyl-2′-O-methyl guanosine bycontacting said N1-benzyl-2′-O-methyl guanosine with sodium naphthaleneunder conditions suitable for the formation of 2′-O-methyl guanosinenucleoside in a crude form; and e) purifying said 2′-O-methyl guanosinenucleoside from said. crude form.

[0035] In one aspect, the invention features a process for the synthesisof 2′-O-methyl adenosine nucleoside, comprising the steps of: a)methylating adenosine by contacting said adenosine withdimethylformamide, magnesium acetylacetonate and trimethylsulphoniumhydroxide under conditions suitable for the formation of 2′-O-methyladenosine in a crude form; and b) purifying said 2′-O-methyl adenosinefrom said crude form.

[0036] In one aspect, the invention features a process for the synthesisof 2′-O-methyl adenosine nucleoside, comprising the steps of: a)methylating adenosine by contacting said adenosine withdimethylformamide, strontium acetylacetonate and trimethylsulphoniumhydroxide under conditions suitable for the formation of 2′-O-methyladenosine in a crude form; and b) purifying said 2′-O-methyl adenosinefrom said crude form.

[0037] In one aspect, the invention features a process for the synthesisof 2′-O-methyl guanosine nucleoside, comprising the steps of: a)contacting 2,6-diaminopurine nucleoside with anhydrous pyridine andTIPSCI under conditions suitable for the formation of2,6-diamino-9-(3′,5′-O-tetraisopropyldisiloxane-(1,3-diyl)-β-D-ribofuranosyl) purine; b)methylating said2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-β-D-ribofuranosyl)purine by contacting said 2,6-Diamino-9-(3′,5′-O-tetraisopropyldisiloxane-(1,3-diyl)-β-D-ribofuranosyl) purine withanhydrous DMF and methyl iodide under conditions suitable for theformation of2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2-O-methyl-β-D-ribofuranosyl)purine; c) acylating said 2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2-O-methyl-β-D-ribofuranosyl)purine by contacting said2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2-O-methyl-β-D-ribofuranosyl) purine with anhydrous pyridine and isobutyrylchloride under conditions suitable for the formation of2,6-Diamino-N²-isobutyryl-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2-O-methyl-β-D-ribofuranosyl)purine; d) deaminating and desilylating said2,6-Diamino-N²-isobutyryl-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2′-O-methyl-β-D-ribofuranosyl)purine under conditions suitable for the formation ofN2-isobutyryl-2′-O-methyl guanosine nucleoside in a crude form; and e)purifying said N2-isobutyryl-2′-O-methyl guanosine nucleoside from saidcrude form.

[0038] In yet another embodiment, the invention features a process forthe synthesis of 2′-O-methyl guanosine nucleoside, comprising the stepsof: a) contacting 2,6-diaminopurine nucleoside with anhydrous pyridineand TIPSCI under conditions suitable for the formation of2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-β-D-ribofuranosyl) purine; b)methylating said2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-β-D-ribofuranosyl)purine by contacting said 2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-β-D-ribofuranosyl) purine withanhydrous DMF and methyl iodide under conditions suitable for theformation of2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2-O-methyl-β-D-ribofuranosyl)purine; c) acylating said 2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2-O-methyl-β-D-ribofuranosyl)purine by contacting said2,6-Diamino-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2′-O-methyl-β-D-ribofuranosyl) purine with anhydrous pyridine andisopropylphenoxyacetyl chloride under conditions suitable for theformation of2,6-Diamino-N²-isopropylphenoxyacetyl-9-(3′,5′-O-tetraisopropyldisiloxane-(1,3-diyl)-2′-O-methyl-β-D-ribofuranosyl) purine; d) deaminating anddesilylating said 2,6-Diamino-N²-isopropylphenoxyacetyl-9-(3,5-O-tetraisopropyldisiloxane-(1,3-diyl)-2-O-methyl-β-D-ribofuranosyl)purine under conditions suitable for the formation ofN²-isopropylphenoxyacetyl-2′-O-methyl guanosine nucleoside in a crudeform; and e) purifying said N²-isopropylphenoxyacetyl-2′-O-methylguanosine nucleoside from said crude form.

[0039] This invention features an improved and economical syntheticmethod for the preparation of 2′-O-methyl nucleosides in high yield. Themethod is not only cost efficient, but can be scaled up to severalhundred gram quantities. The method generally utilizes inexpensivecommercially available 2,2′-anhydro-1(β-D-arabinofuranosyl) nucleoside,such as 2,2′-anhydro-1(β-D -arabinofuranosyl)uracil or2,2′-anhydro-1(β-D-arabinofuranosyl)cytosine, as a starting materialwhich is converted in a one or two step reaction sequence to 2′-O-methylnucleosides with a yield of about 90-100%.

[0040] The 2′-O-methyl or 3′-O-methyl nucleosides can be used forchemical synthesis of nucleotides, nucleotide-tri-phosphates and/orphosphoramidites as a building block for selective incorporation intooligonucleotides. These oligonucleotides can be used as an antisensemolecule, 2-5A antisense chimera, triplex molecule or as an enzymaticnucleic acid molecule. The oligonucleotides can also be used as probesor primers for synthesis and/or sequencing of RNA or DNA.

[0041] By “antisense” it is meant a non-enzymatic nucleic acid moleculethat binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA(protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactionsand alters the activity of the target RNA (for a review see Stein andCheng, 1993 Science 261, 1004).

[0042] By “2-5A antisense chimera” it is meant, an antisenseoligonucleotide containing a 5′ phosphorylated 2′-5′-linked adenylateresidues. These chimeras bind to target RNA in a sequence-specificmanner and activate a cellular 2-5A-dependent ribonuclease which, inturn, cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad.Sci. USA 90, 1300).

[0043] By “triplex DNA” it is meant an oligonucleotide that can bind toa double-stranded DNA in a sequence-specific manner to form atriple-strand helix. Formation of such triple helix structure has beenshown to inhibit transcription of the targeted gene (Duval-Valentin etal., 1992 Proc. Natl. Acad. Sci. USA 89, 504).

[0044] By “enzymatic nucleic acid” it is meant a nucleic acid moleculecapable of catalyzing reactions including, but not limited to,site-specific cleavage and/or ligation of other nucleic acid molecules,cleavage of peptide and amide bonds, and trans-splicing.

[0045] In preferred embodiments, the invention features a method forchemical synthesis of 2′-O-methyl or 3′-O-methyl nucleosides in which2,2′-anhydro-1(β-D-arabinofuranosyl) cytosine,2,2′-anhydro-1(β-D-arabinofuranosyl) uracil,2,3′-anhydro-1(β-D-arabinofuranosyl) uracil, or2,3′-anhydro-1(β-D-arabinofuranosyl) cytosine is used as the startingmaterial, and wherein said starting material is reacted with a Lewisacid.

[0046] Another preferred embodiment of the invention features a methodfor chemical synthesis of 5′-O-methylpyrimidine nucleoside in which2,5′-anhydro-1(β-D-arabinofuranosyl) pyrimidine is used as the startingmaterial and is reacted with a Lewis acid.

[0047] In yet another preferred embodiment, the invention features novelprocesses for the synthesis of 2′-O-methyl purine nucleosides.

[0048] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] The drawing will first briefly be described.

[0050] Drawing:

[0051]FIG. 1 is a diagrammatic representation of a scheme involved inthe synthesis of 2,2′-anhydro-1 (β-D-arabinofuranosyl) uracil (2).

[0052]FIG. 2 is a diagrammatic representation of a scheme involved inthe synthesis of 2′-O-methyl uridine (3) by the method of thisinvention.

[0053]FIG. 3 is a diagrammatic representation of a scheme involved inthe synthesis of 2′-O-methyl cytidine (5) by the method of thisinvention.

[0054]FIG. 4 is a diagrammatic representation of a scheme involved inthe synthesis of 3′-O-methylpyrimidine nucleosides.

[0055]FIG. 5 is a diagrammatic representation of a scheme involved inthe synthesis of 5′-O-methylpyrimidine nucleosides.

[0056]FIG. 6 shows NMR profile of 2′-O-methyl nucleosides. A)2′-O-methyl Uridine nucleoside. B) 2′-O-methyl cytidine nucleoside.

[0057]FIG. 7 is a diagrammatic representation of a scheme for thesynthesis of 2′-O-methyl adenosine nucleoside.

[0058]FIG. 8 is a diagrammatic representation of a scheme for thesynthesis of 2′-O-methyl adenosine and guanosine nucleosides.

[0059]FIG. 9 is a diagrammatic representation of a scheme for thesynthesis of 2′-O-methyl guanosine nucleoside.

[0060]FIG. 10 is a diagrammatic representation of a scheme for thesynthesis of methoxy nucleosides via N1-Benzyl guanosine route.

EXAMPLE 1 Synthesis of 2.2′-Anhydro-1(β-D-Arabinofuranosyl) Uracil (2)

[0061] 2,2′-anhydro-1(β-D-arabinofuranosyl) uracil (2) can either bepurchased from Sigma Chemicals or can be synthesized using the schemedescribed by Verheyden et al., 1971 (J. Org. Chem. 36, 250). Briefly, toan oven baked 1L 3-neck round bottom flask equipped with mechanicalstirrer, reflux condenser, and positive pressure of argon, 200 g (0.819mol) of uridine (1) was added. The reaction was carried out in thepresence of diphenylcarbonate (191.2 g, 0.9 mol), and DMF (300 ml). Theresulting light yellow suspension was heated to 90° C. at which timesodium bicarbonate (2.0 g) was added. The reaction mixture was thenheated to 110° C. for two hours during which time CO₂ evolved. Over thistwo hour period, the reaction mixture transformed from a slurry to ahomogeneous solution and back to a slurry. Upon cooling to −10° C., thereaction mixture was filtered and the filter bed washed with ethanol andcold methanol. The filter bed was then suspended in methanol (500 ml)and heated to reflux for three hours. After cooling to −10° C., thereaction mixture was filtered. The filter bed was washed with coldmethanol and dried to retrieve 2 as an off white solid (140 g; 76%).

EXAMPLE 2 Synthesis of 2′-O-Methyl Uridine (3)

[0062] To an oven baked stainless steel bomb (300 ml), equipped withmagnetic stirrer and purged with argon, 40 ml anhydrous methanol wasadded followed by the addition of2,2′-anhydro-1-(β-D-arabinofuranosyl)uracil 2 (1.0 g, 4.42 mmol). To theresulting slurry, 5.0 ml trimethylborate (44.2 mmol) was added followedby the addition of boron trifluoride-methanol (50%) (1.5 ml, 8.84 mmol).The bomb was then sealed and heated in an oil bath at 130° C. for 18hours. Upon cooling, the resulting clear, slightly colored reactionmixture was evaporated in vacuo to yield a dark foam. The crude foam wasdissolved in minimal methanol/dichloromethane and applied to a flashsilica gel column. A gradient of 10-30% EtOH in dichloromethane afforded3 as a white foam (1.05 g, 92%).

[0063] Alternately, to an oven baked stainless steel bomb (920 ml),equipped with magnetic stirrer and purged with argon, 200 ml anhydrousmethanol was added followed by the addition of 2 (50 g, 0.221 mol).Trimethylborate (400 ml, 3.54 mol) was added to the resulting slurry andthe bomb was sealed. The bomb was then heated in an oil bath at 130° C.for 38 hours. Upon cooling, the resulting clear, slightly coloredreaction mixture was evaporated in vacuo to afford an off white foam.Crystallization of the crude product from (methanol/ethyl acetate) gavepure 3 (56.8 g, 100%).

[0064] The identity and purity of the synthesized compound was confirmedby standard NMR analysis (FIG. 6A). Following is the result of NMRanalysis: ¹H NMR DMSO: 11.33 (exch. s, 1H, NH), 7.92 (d, J_(6,5)=8.2,1H, H6), 5.85 (d, J_(1′,2′)=5.2, 1H, H1′), 5.65 (d, J_(5,6)=8.2, 1H,H5), 5.13 (exch. m, 2H, 5′OH, 3′OH), 4.10 (t, J_(3′, 2′)=4.9,J_(3′,4′)=4.6, 1H, H3′), 3.85 (m, 1H, H4′), 3.62 (dd, J_(5′, 4)′=3.0,J_(5′, 5)′=12.1, 1H, H5′), 3.54 (dd, J_(5″, 4′)=3.1, J_(5″, 5′)=12.1,1H, H5″), 3.35 (s, 3H, OCH₃). The peak corresponding to the 2′-O-methylis indicated in the FIG. 6A.

EXAMPLE 3 2′-O-methyl cytidine (5)

[0065] To an oven baked stainless steel bomb (300 mL), equipped withmagnetic stirrer and purged with argon, 50 ml anhydrous methanol wasadded followed by the addition of commercially available 1 g2,2′-Anhydro-1-(β-D -arabinofuranosyl)cytosineacetate (Aldrich) 4 (3.5mmol). To the resulting slurry, 8 ml Trimethylborate (70 mmol) was addedin the presence or absence of boron trifluoride-methanol (50%) (1.5 ml,8.84 mmol). The bomb was sealed and then heated in an oil bath at 130°C. for 38-48 hours. Upon cooling, the resulting clear, slightly coloredreaction mixture was evaporated in vacuo to afford an off white foam.After drying in vacuo, the crude foam was dissolved in anhydrous DMF (50ml) and acetic anhydride (0.36 ml, 3.85 mmol) which was added drop-wiseto the reaction mixture. The resulting clear, light yellow solution wasstirred overnight at room temperature. The reaction mixture wasevaporated in vacuo. Crystallization of the crude product from(methanol/ethyl acetate) gave a pure compound 5 (0.94 g, 90%).

[0066] The identity and purity of the synthesized compound was confirmedby standard NMR analysis (FIG. 6B). Following is the result of NMRanalysis: ¹H NMR DMSO: 10.89 (exch. s, 1H, NH), 8.46 (d, J_(6,5)=7.4,1H, H6), 7.18 (d, J_(5,6)=7.4, 1H, H5), 5.83 (d, J_(1′,2′)=2.5, 1H,H1′), 5.18 (exch. t, J_(OH, 5)=4.6 J_(OH, 5″)=4.9, 1H, 5′OH), 5.08(exch. d, J_(OH,3)=6.7, 1H, 3′OH), 4.04 (t, J_(3′,2′)=4.9,J_(3′,4′)=6.8, 1H3′), 3.88 (m, 1H, H4′), 3.75 (dd, J_(5′,4′)=2.3,J_(5′,5″)=12.2, 1H, H5′), 3.59 (dd, J_(5″,4′)=2.5, J_(5″,5′)=12.2, 1H,H5″), 3.45 (s, 3H, OCH₃), 2.10 (s, 3H, CH₃).

[0067] 2′-O-methyl nucleosides of the present invention can be readilyconverted into phosphoramidites using standard procedures andphosphoramidites can be readily incorporated into oligonucleotides, suchas RNA, using standard procedures described in Sproat & Gait, 1984 inOligonucleotide Synthesis: A Practical Approach, ed. Gait, M. J. (IRL,Oxford), pp 83-115; Usman et al., 1987 J. Am. Chem. Soc., 109, 7845;Scaringe et al., 1990 Nucleic Acids Res., 18, 5433; and Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684.

EXAMPLE 4 Synthesis of 3′-O-methyl Pyrimidine Nucleoside

[0068] Referring to FIG. 4, the treatment of 2,3′-anhydro-1-(β-D-arabinofuranosyl)primidine 6 (Aldrich) with anhydrous methanol andtrimethylborate, in the presence or absence of borontrifluoride-methanol, in an oven baked stainless steel bomb purged withargon, followed by heating in an oil bath at 130° C. for 18-48 hours, asdescribed above, will yield 3′-O-methyl pyrimidine nucleoside 7.

EXAMPLE 5 Synthesis of 5′-O-methyl Pyrimidine Nucleoside

[0069] Referring to FIG. 5, the treatment of 2,5′—anhydro-1-(β-D-arabinofuranosyl)primidine 8 (Aldrich) with anhydrous methanol andtrimethylborate, in the presence or absence of borontrifluoride-methanol, in an oven baked stainless steel bomb which ispurged-with argon, followed by heating in an oil bath at 130° C. for18-48 hours, as described above, will yield 5′-O-methylpyrimidinenucleoside 9.

EXAMPLE 6 Synthesis of 2′-O-Me -Adenosine via Transglycosilation (Scheme1: FIG. 7)

[0070] In 1982, Imbach et al., (J. Org. Chem. 1982, 47, 202)demonstrated utilization of2′-O-methyl-1,3,5-tri-O-benzoyl-α-D-ribofuranose in the stereospecificsynthesis of 2′-O-methylpyrimidine-β-D-ribonucleoside by theglycosilation of silylated bases, using Lewis acid as a catalyst. Thisprocedure was optimized for large scale preparation of2′-O-methylpyrimidine ribonucleosides by Ross et al., (J. HetercyclicChem. 31,765 1994). This procedure required methylation of1,3,5-tri-O-benzoyl-α-D-ribofuranose with a large excess of potentiallyexplosive diazomethane.

[0071] As an alternate, Applicant describes transglycosilation ofsuitably protected 2′-O-Me-pyrimidine ribonucleosides obtained throughB(OMe)₃ mediated opening of 2,2′-anhydronycleosides. Transglycosylationreaction proceeds usually with high β-selectivity, if the carbohydratedonor contains a 2′-O-acyl group capable of stabilising the postulatedC-1 carboxonium ion for exclusive β— attack by the incoming silylatedbase. The stereochemical outcome of transglycosilation reaction withcarbohydrate donor, without the participating group at 2′-position (as2′-O-Methyl) usually results in a mixture of α, and β nucleosides, asdocumented for the synthesis of 2′-N₃ purine ribonucleoside (Imazawa andEckstein, 1979, J.Org. Chem. 44, 2039-41).

[0072] Applicant has investigated transglycosilation of5′,3′-di-O-acetyl-2′-O-Methyl uridine, obtained by the acylation of2′-O-Methyl uridine. The transglycosilation of5′,3′-di-O-acetyl-2′-O-Methyl uridine with 3 eq of N⁶-benzoylaminopurineand 3 eq TMStriflate in CH₃CN at 75° C. for 16 hour resulted inunseparable mixture of α and β isomers of N⁶-Benzoyl-5′,3′-di-O-acetyl-2′-O-methyl adenosine in -60% yield and 1:1 ratio. Since thenature of aglycone can also influence product distribution intransglycosilation reaction (Azuma and Isono, 1977, Chem. Pharm. Bull.25, 3347-53), we decided to try N⁴-acetyl-5′,3′-di-O-acetyl-2′-O-methylcytidine (2) as a carbohydrate donor. Suprisingly, utilization of thisdonor under the same conditions as described above for 2′-O-Methyluridine derivative resulted in the exclusive formation of β anomer ofN⁶-Benzoyl-5′,3′-di-O-acetyl-2′-O-methyl adenosine in 50% yield. WhenN⁶-phenoxyacetylaminopurine was used instead of N⁶-Benzoyl-adenine underthe same conditions described above, extensive decomposition ofinitially formed adenosine derivative was observed and target nucleosidewas not isolated.

EXAMPLE 7 Synthesis of 2′-O-Methyl-Guanosine and Adenosine from2-amino-6-Chloropurine Riboside (Scheme 2: FIG. 8).

[0073] High regioselectivity in methylation of 6-Cl-guanosine bydiazomethane was reported by Robins' laboratory in 1966 (Khawaia andRobins, J. Am. Chem. Soc. 1966, 88 ,3640-43). Despite the exclusivemethylation of 2′-OH, the 2′-O-Methyl-6-CI-guanosine was not isolated.Subsequent transformation of this key intermediate resulted in thepreparation of 2′-O-methyl-guanosine in 30% yield. Moreover, the use ofdiazomethane as a methylation agent is not practical for large scalepreparations. It was therefore reasonable to investigate othermethylation reagents.

[0074] Suprisingly, Applicant has found that the methylation of2-amino-6-chloropurine riboside with a small excees of NaH/Mel reagentin CH₂Cl₂ at −20° C. resulted in 2′-O-Me-6-Cl-guanosine in 65% yieldalong with the formation of 2′,3′ bis-O-Me derivative in 15% yield; no3′-methylation was observed under these conditions. Several procedureswere tested for the transformation of intermediate (5) into2′-O-Methyl-guanosine (5). Applicant found that, the best result wasobtained when intermediate (5) was treated with1,4-diazabicyclo[2.2.2]octane (1 equiv.) and water (30 mL) at 90° C. for45 minutes, followed by hydrolysis with 2M NaOH at pH 12 (FIG. 8). Thedesired 2′-O-Methyl-guanosine was obtained in 65% yield.

[0075] The surprisingly high regio-selectivity observed in themethylation of 2-amino-6-chloropurine riboside facilitates large scalesynthesis of 2′-O-Me-6-Cl-guanosine, which can serve as a keyintermediate in the preparation of not only 2′-O-Methyl-guanosine, butalso 2′-O-Methyl-adenosine. This latter transformation was achievedthrough radical deamination (Nair and Richardson, Synthesis, 1982670-672) of 3′,5′-di-O-Acetyl-2′-O-Methyl-6-Chloro-2-aminopurineriboside (6) which yields 3′,5′-di-O-Acetyl-2′-O-Methyl-6-Chloropurine(7) in 72% yield starting from 5. Subsequent amination of (7) withmethanolic ammonia at 125° C. for 4 hours yields 2′-O-Methyl-adenosinein 80% yield.

EXAMPLE 8 Synthesis of N²-Acyl(Isobutyryl and Isopropylphenoxyacetyl)-2′-O-Methylguanosine from 2,6-Diamino-β-D-Ribofuranosylpurine (Scheme3: FIG. 9)

[0076] It has been reported that the diazomethane methylation of2,6-Diamino -β-D-ribofuranosylpurine in the presence of SnCl₂×2H₂Oprovided a 1:1 mixture of 2′- and 3′-O-methylated derivatives in aquantitative yield. These compounds can be separated on Dowex 1 OHcolumn and deaminated to the corresponding Guanosine derivatives withAdenosine Deaminase (Robins et al., Can. J. Chem. 1981, 59, 3360). It isalso known in the literature that direct methylation of guanosineusually resulted in preferential methylation of N₁ and/or N₇ positionsof the base. In 2,6-Diamino-β-D-ribofuranosylpurine, the acidic amidefunction C6-N1— is replaced by an basic amidine function, therefore onewould expect that such a replacement should reduce the extent ofmethylation at N1 under basic conditions (i.e. NaH/Mel). In order toincrease the regioselectivity, Applicant used5′,3′-O-tetraisopropyldisiloxane-1,3-diyl protection which has beenreported to be relatively stable under NaH/Mel methylation conditions(Parmentier et al., 1994, Tetrahedron, 50, 5361-68).

[0077] The 5′,3′-O-tetraisopropyldisiloxane-1,3-diyl protection wasintroduced by standard procedure, utilization of ethylacetate-waterextraction allowed isolation of pure protected derivative (11) in 90%yield in crystalline form due to low solubility in above system.

[0078] Applicant has investigated several methylation procedures for theNaH/Mel system, including, varying the amount and the type of solventand equivalents of NaH and Mel. Best results were obtained whenmethylation was performed at O° C. in DMF with 1.5 eq of NaH and 3 eq.of Mel. If the reaction is performed at higher temperatures or in a moreconcentrated solution, extensive hyper-methylation occured. Utilizationof lower amounts of Mel required longer reaction time and resulted inthe opening of cyclic silyl group with the simultaneous methylation ofboth 2′ and 3′ hydroxyl groups. The 2′-O-Methyl derivative 12 can thenbe isolated in 90% yield by crystallization and without any columnchromatography (FIG. 9).

[0079] In order to synthesize N²-acyl -2′-O-methylguanosine derivatives14 and 15, Applicant tested selective acylation of N² amino group ofintermediate 12 with subsequent chemical deamination of N⁶ amino group.Two factors are critical for the success of this approach: the degree ofselectivity in acylation of N² amino group vs N⁶ amino group inintermediate 12 and the stability of N² protection under acidicconditions of deamination (Davoll et al., J Am. Chem. Soc. 1951, 73,1650). Applicant has found that when the acylation of diamonopurine 12is performed at −10° C. with 1.1 eq of acyl chloride, exclusiveacylation of N² amino group in 12 occured (FIG. 9). For example, theN²-isobutyryl intermediate 13 was isolated in 97% yield. The structureof 13 was confirmed by NMR data and by deamination to N²-isobutyryl-2′-O-methylguanosine 14 with NaNO₂/CH₃COOH followed by desilylation withTEA3HF. Applicant also observed that during deamination withNaNO₂/CH₃COOH, 3′,5′-O-cyclic silyl group opened, presumably at the5′-position. This intermediate was not isolated but desilylated directlywith TEA3HF. It is worth noting that N²-isobutyryl group was completelystable during deamination. The presence of hydrophobic silyl group inthe intermediate allowed easy separation by extraction from excess ofinorganic salts which made possible the subsequent desilylation withoutisolation.

[0080] High yields obtained in all the steps of transformation from 12to 14 prompted us to combine these reactions in a “one pot” procedurewithout the isolation of intermediate 13. Selective acylation of 12 withisobutyryl chloride followed by deamination with NaNO₂/CH₃COOH anddesylilation with TEA3HF resulted in N²-isobutyryl-2′-O-methylguanosine14 in 88% yield.

[0081] We also applied this procedure for the synthesis ofN²-isopropylphenoxyacetyl-2′-O-methylguanosine on a 50 g scale. TheN²-isopropylphenoxyacetyl protection was also stable under acidicdeazotation conditions, athough a minor loss of this group was observedresulting in a 83% overall yield starting from 12.

EXAMPLE 9 Synthesis of 2′-O-Methylguanosine and 2′-O-Methyl-adenosinevia Metal-Directed Methylation (Scheme 4: FIG. 10 and Table II)

[0082] It has been demonstrated that metal acetylacetonates can directmethylation of ribonucleosides with trimethylsulfonium hydroxide, mostlyat the 2′ and 3′ hydroxyl groups of Uridine, Cytidine and Adenosine(Yamauchi et al., J. Org Chem. 1980, 45, 3865-68). Application of thisprocedure to the synthesis of Guanosine derivatives resulted in theisolation of 6 compounds with methylation in the base and the ribosemoeties. When N¹-methyl-guanosine was subjected to the same methylationconditions 1,2′ and 1,3′ di-N-O methyl guanosines were isolated in 82%yield.

[0083] Applicant investigated procedures for metal-directed methylationof N¹-protected Guanosine with trimethylsulfonium hydroxide to optimizethe ratio of 2′:3′ methylated products with subsequent separation anddebloking to obtain 2′-O-Methyl guanosine.

[0084] The protection of N¹ in guanosine was achieved using N¹benzylation with N,N-dimethylformamide dibenzyl acetal (Philips andHorwitz J. Org. Chem. 1975, 40,1856). Applicant observed that completecleavage of 2′,3′-orthoamide required more drastic conditions thanpreviously reported (2N NaOH vs MeOH/NH₃) (FIG. 10). The target compoundwas isolated in 80% yield after crystallization.

[0085] Several metal acetylacetonates were tested in methylationreaction (See Table 1). Whereas Cu²⁺ acetylacetonate mediatedmethylation produced 1:1 ratio of 2′- and 3′-O-methylated products;Mg²⁺and Ag⁺directed methylation changed the ratio to 9:1. With Ag⁺theoverall conversion was higer than with Mg²⁺ resulting in a 70% isolatedyield of 2′-O-Me-N₁-BzI-guanosine (FIG. 10; 17). The separation of2′-O-and 3′-O-Me-N₁-Bzl-guanosine derivative was achieved on apreparative scale on Waters Delta-Pak ODS 50 mm×300 mm HPLC column.Removal of N₁-BzI protection with Na⁺naphtalene provided2′-O-Me-Guanosine in 90% yield.

[0086] Applicant investigated 12 different acetylacetonates in thedirect methylation of Adenosine (Table II). Whereas Fe and Cuacetylacetonates provided 1:1 and 2:1 ration of 2′ and 3′ isomers asreported, Applicant discovered that Ag⁺ and Sr²⁺ allows equilibrium toshift towards 2′-isomer, providing 4:1 and 8:1 ratio. This allows theisolation of 2′-O-Me adenosine in 75-80% yield.

Experimental Procedures for the Synthesis of 2′-O-Methyl Adenosine andGuanosine Nucleosides

[0087] NMR spectra were recorded on a Varian Gemini 400 spectrometeroperating at 400.075 MHz for proton and 161.947 MHz for phosphorus.Chemical shifts in ppm refer to TMS and H₃PO₄, respectively. Analyticalthin-layer chromatography (TLC) was performed with Whatman MK6F silicagel 60 Å F₂₅₄ plates and column chromatography using Merck 0.040-0.063mm Silica gel 60.

[0088] N⁶Benzoyl-5′,3′-di-O-Acetyl-2′-O-Methyl Adenosine (3):

[0089] To a solution of N⁴-acetyl-2′-O-methyl cytidine(1) (1.87 g, 6.25mmol) stirring at RT under argon in DMF/pyridine (20 ml, 20 ml) wasadded acetic anhydride (1.76 ml, 18.75 mmol) via syringe. The reactionmixture was stirred at RT for 18 hours then quenched with EtOH (2 ml).The reaction mixture was evaporated to dryness in vacuo and partitionedbetween dichloromethane and sat. NaHCO₃. The aqueous layer was backextracted with additional dichloromethane and the combined organicsdried over Na₂SO₄. After filtration, the filtrate was-evaporated invacuo to afford a white foam.

[0090] A solution of N⁶-benzoylaminopurine (Lancaster) (1.23 g, 5.16mmol) stirring in anhydrous acetonitrile under an argon atmosphere wastreated with BSA (3.82 ml, 15.48 mmol) at reflux for 3 hours. Uponcooling, a solution of N⁴-acetyl-5′,3′-di-O-acetyl-2′-O-methyl cytidine(2) (see above) (0.66 g, 1.72 mmol) in 20 ml anh. acetonitrile was addedto the reaction mixture followed by. TMStriflate (1.03 ml, 5.16 mmol).The reaction mixture was then heated to 75° C. for 16 hours whilestirring under positive pressure argon. Upon cooling, an additional 1.03ml (5.16 mmol) of TMStriflate was added, and the reaction heated to 75°C. for an additional 20 hours. Once cool, the reaction mixture wasdiluted with two volumes of dichloromethane and washed with sat. NaHCO₃.The organic layer was then dried over Na₂SO₄ and evaporated in vacuo.Flash chromatography employing a gradient of 10 to 80% ethylacetate/hexanes afforded (1) as a white foam; 0.403 g, 50% yield. ¹H NMR(CDCl₃): 8.88 (br s, 1H, NH), 8.81 (s, 1H, H8), 8.31 (s, 1H, H2),8.11-7.53 (m, 5H, benzoyl), 6.18 (d, J_(1′,2)=4.8, 1H, H1′), 5.41 (t,J_(3′,2′)=4.8, J_(3′,4′)=4.8, 1H, H3′), 4.75 (t, J_(2′,1′)=4.8,J_(2′,3′)=4.8, 1H, H2′), 4.50-4.34 (m, 3H, H4′, H5′, H5″), 3.44 (s, 3H,OCH₃), 2.19 (s, 3H, OAc), 2.14 (s, 3H, OAc).

[0091] 2′-O-Methyl-2-Amino-6-Chloropurine Riboside (5)

[0092] Sodium hydride. (0.44 g, 18.2 mmol) was added to the cooled (−20°C.) solution of 2-amino-6-chloropurine riboside 4 (5 g, 16.6 mmol) indry dimethylformamide (100 mL) under stirring. After 1 hour the solutionof CH₃l (1.24 mL, 19.9 mmol) in dry dichloromethane (10 mL) was addeddropwise to the reaction mixture during 1 hour. Resulted yellow solutionwas stirred at −20° C. for additional 2 hours until TLC (methylenechloride-methanol 9:1) showed complete disappearance of startingmaterial. Reaction mixture was quenched with methanol (20 mL), warmed tothe room temperature and evaporated to dryness in vacuo. The residue wasdissolved in water (200 mL) and extracted with methylene chloride (3×200mL). Organic layer was back extracted with water (100 mL). Combinedaqueous phase was evaporated to dryness and the residue was purified byflash chromatography on silica using gradient of MeOH (7% to 10%) inmethylene chloride to give 3.4 g (65%) of the compound 5. Flashchromatography purification can be substituted by multiplecrystallization from acetonitrile, m.p. Calcd. for C₁₀H₁₂N₅O₄Cl(301.69): C 39.81; H 4.01; N 23.21; Cl 11.75 found C ; H ; N ; Cl.¹H-NMR (DMSO-d₆): 3.32 (3H, s, 2′-OMe); 3.578 (1H, dd, 5′-H,J_(4′,5′)4.0, J_(5′,5′)12.0); 3.66 (1H, dd, 5′-H, J_(4′,5′)4.0,J_(5′,5′)12.0); 3.949 (1H, dd, 4′-H, J_(3′,4′)3.6); 4.252 (1H, t,2′-H,J_(2′,3′)4.0); 4.312 (1H, m, 3′-H); 5.073 (1H, bs, 3′-OH, exchangeable);5.236 (1H, bs, 5′-OH, exchang-eable); 5.908 (1H, d, 1′-H, J_(1′,2)′6.0);6.964 (2H, bs, 2-NH₂); 8.391 (1H, s, 8-H).

[0093] 2′-O-Methyl Guanosine (9)

[0094] A mixture of 5 (2.05 g, 6.5 mmol), 1,4-diazabicyclo[2.2.2]octane(1 equiv.) and water (30 mL) was heated to 90° C. for 45 minutes. Thenthe solution was cooled to ambient temperature, basified to pH 12 with2M NaOH, and washed with methylene chloride (3×60 mL). The aqueous phasewas acidified to pH 6 with 6M HCl and left at refrigerator overnight.Formed precipitate was filtered off. Mother liquor was evaporated todryness, the residue was dissolved in water and applied to the shortcolumn with RP-18 silica gel. Solid phase was washed with water andremaining product was eluted with 5% aq methanol. Appropriate fractionswere combined, evaporated to dryness and recrystallized from to provide1.25 g (65%) of 2′-O-methyl guanosine 9. Analytical sample wasrecrystalized from water, m.p. The product was identical to authenticsample by HPLC, UV, ¹H-NMR-spectroscopy.

[0095] 3′5′-Di-O-Acetyl-2′-O-Methyl-6-Chloropurine Riboside (7)

[0096] To the solution of compound 5 (1.25 g, 3.96 mmol),4-dimethylaminopyridine (39 mg, 0.32 mmol) and triethylamine (0.37 mL,2.64 mmol) in dry acetonitrile was added acetic anhydride (0.9 mL, 9.5mmol) and the reaction mixture was left at room temperature for 40minutes. Then it was quenched with MeOH (10 mL) and evaporated todryness. The residue was dissolved in methylene chloride and washed with1% aq acetic acid, saturated aq sodium bicarbonate, and brine. Theorganic layer was dried over sodium sulfate and evaporated to drynessyielding acetate 6. The residue was additionally dried in vacuo for 3hours, dissolved in dry THF and degassed with dry argon. To the aboveboiling solution under positive pressure of argon isoamyinitrite (10 eq)was added dropwise. After 2 hours solvent was removed in vacuo and theresidue was dissolved in methylene chloride, washed with saturated aqNaHCO₃ and brine. The residue after evaporation of organic phase waspurified by flash chromatography on silica gel. Elution withhexanes-ethyl acetate (1:1) mixture provided 1.1 g (72%) of compound 7as yellow oil. Calcd. for C₁₅H₁₇N₄O₆Cl (384.78): C 46.82; H 4.45; N14.56; Cl 9.21; found C ; H ; N ; Cl ¹H-NMR (CHCI₃-d): δ2.133 (3H, s,3′-OAc or 5′-OAc); 2.184 (3H, s, 3′-OAc or 5′-OAc); 3.44 (3H, s,2′-OCH₃); 4.425 (3H, m, 4′-H, 5′-CH₂); 4.686 (1 H, t, 2′-H,J_(2′,3′)5.04); 5.36 (1H, t, 3′-H, J_(3′,4′)4.28); 6.132 (1H, d, 1′-H,J_(1′,2′)4.88); 8.311 (1H, s, 8-H); 8.771(1H, s, 2-H).

[0097] 2′-O-Methyl Adenosine (8)

[0098] Solution of compound 7 (0.45 g, 1.17 mmol) in saturatedmethanolic ammonia (20 mL) was autoclaved at 125° C. for 4 hours. Thesolvent was removed in vacuo and remaining residue was purified by flashchromatography on silica gel. Elution with methylene chloride—methanol(9:1) mixture provided 0.25 g (80%) of 2′-O-methyl adenosine 8 as whitesolid. The analytical sample was recrystallized from abs EtOH. m.p. Theproduct was identical to authentic sample by HPLC, UV-, ¹H-NMR-spectroscopy.

[0099]2,6-Diamino-9-[3′,5′-O-Tetraisopropyldisiloxane-1,3-diyl)-β-D-Ribofuranosyl]Purine(11):

[0100] To an oven baked 500 ml three neck round bottom flask equvipedwith mechanical stirrer, positive pressure argon, and rubber septum wasadded 2,6-Diamino-9-(β-D-ribofuranosyl)purine (10) (10.0 g, 35.4 mmol),anhydrous DMF (100 ml), and anhydrous pyridine (200 ml). The resultinglight brown suspension was cooled to 0° C. in an ice/water bath whilestirring. TIPSCl (42.48 mmol, 13.6 ml) was added dropwise to the stirred0° C. reaction mixture via syringe over a 20 minute period. The reactionmixture was then warmed to rt resulting in a homogenious solution. TLCindicated complete reaction after 3 hours at rt, at which time thereaction was quenched by addition of ethanol (20 ml). The reactionmixture was then evaporated in vacuo and the resulting residuepartitioned between ethyl -acetate and sat. aqueous NaHCO₃ at which time(11) precipitated from the organic layer. The aqueous layer was thenback extracted with ethyl acetate and the combined orgainics cooled to0° C. The precipitate was filtered and washed with ethyl acetate toafford (11) as a beige solid; 16.5 g, 89% yield. ¹H NMR (dmso-d6): 7.77(s, 1H, H8), 6.75 (s, exch, 2H, N⁶—NH₂), 5.74 (s, exch, 2H, N²—NH₂),5.71 (s, 1H, H1′), 5.56 (d, J_(OH,2)=5.0, 1H, 2′-OH), 4.43 (dd,J_(3′,2′)=4.5, J_(3′,4′)=7.8, 1H, H3′), 4.29 (m, J_(2′,OH)=5.0),4.06-3.88 (m, 3H, H4′, H5′, H5″), 1.04 (m, 28H, TIPDS).

[0101]2,6-Diamino-9-[3′,5′-O-Tetraisopropyldisiloxane-1,3-Diyl)-2′-O-Methyl-β-D-Ribofuranosyl]Purine(12):

[0102] To an oven baked 500 ml three neck round bottom flask equipedwith mechanical stirrer and positive pressure argon was added (11) (15.6g, 29.7 mmol) followed by anhydrous DMF (300 ml) and methyl iodide (89.2mmol, 5.55 ml). The reaction mixture was cooled to 0° C. in an ice/waterbath and 60% sodium hydride in oil (44.6 mmol, 1.78 g) added slowly. Atemperature of 0° C. was maintained for 35 minutes at which time thereaction was quenched with anhydrous ethanol and diluted into 2 volumesof 0° C. dichloromethane. The dilute reaction mixture was washed twotimes with sat. NH₄CI, the aqueous layer back extracted withdicloromethane, and the combined organics dried over Na₂SO₄, filteredand evaporated to dryness in vacuo. Crystallization from ethanol/water1:1 afforded 14.7 grams of (12), 92% yield. ¹H NMR (dmso-d6): 7.75 (s,1H, H8), 6.76 (s, exch, 2H, N⁶—NH₂), 5.78 (s, 1H, H1′), 5.73 (s, exch,2H, N²—NH₂), 4.58 (dd, J_(3′,2′)=4.8, J_(3′,4′)=4.8, 1H, H3′), 4.12 (d,J_(2′,3′)=4.8), 4.09-3.91 (m, 3H, H4′, H5′, H5″), 3.54 (s, 3H, OCH₃),1.03 (m, 28H, TIPDS).

[0103]2,6-Diamino-N²-Isobutyryl-9-[3′,5′-O-Tetraisopropyldisiloxane-1,3-Diyl)-2′-O-Methyl-β-D-Ribofuranosyl]Purine (13):

[0104] A solution of (3) (0.5 g, 0.93 mmol) in anhydrous pyridine (20ml) was cooled to −10° C. in an ice/ethanol bath while stirring underargon. Isobutyryl chloride (1.02 mmol, 0.11 ml) was added dropwise tothe stirred −10° C. solution over a period of 5 minutes. The reactionmixture was stirred at −10° C. for 2 hours followed by 1 hour at rt thenquenched with ethanol (2 ml). After evaporating the reaction mixture todryness in vacuo, the resulting residue was partitioned betweendichloromethane and sat. aqueous NaHCO₃. The aqueous layer was backextracted with dichloromethane and the combined organics dried overNa₂SO₄. Filtration and evaporation of the filtrate in vacuo afforded abeige-foam. Flash chromatography using a gradient of 2-4% ethanol indichloromethane afforded (13) as a white foam; 0.55 g, 97% yield. ¹H NMR(dmso-d6): 9.76 (s, exch, 1H, N²—NH), 8.04 (s, 1H, H8), 7.20 (s, exch,2H, N⁶—NH₂), 5.88 (s, 1H, H1′), 4.71 (dd, J_(3′,2′)=5.2, J_(3′,4′)=5.2,1H, H3′), 4.26 (d, J_(2′,3′)=5.2), 4.15-3.91 (m, 3H, H4′, H5′, H5″),3.55 (s, 3H, OCH₃), 2.87 (m, 1H, iBu—CH), 1.06-0.96 (m, 34H, TIPDS,iBu—(CH₃)₂).

[0105] N²-Isobutylryl-2′-O-Methylguanosine (14):

[0106] A solution of (13) (5.0 g, 9.28 mmol) in anhydrous pyridine (100ml) was cooled to −10° C. in an ice/ethanol bath while stirring underargon. Isobutyryl chloride (10.21 mmol, 1.07 ml) was added dropwise tothe stirred −10° C. solution over a period of 30 minutes. The reactionmixture was stirred at −10° C. for 2 hours followed by 1 hour at rt thenquenched with ethanol (20 ml). After evaporating the reaction mixture todryness in vacuo, the resulting residue was partitioned betweendichloromethane and sat. NaHCO₃. The aqueous layer was back extractedwith dichloromethane and the combined organics dried over Na₂SO₄.Filtration and evaporation of the filtrate in vacuo afforded a beigefoam which was dissolved in glacial acetic acid (80 ml). To the stirredacetic acid solution was added water (40 ml) followed by NaNO₂ (74.24mmol, 5.12 g). Another portion of NaNO₂ (74.24 mmol, 5.12 g) was addedafter 30 minutes and the reaction stirred at rt for 48 hours. Thereaction mixture was diluted with one volume of n-butanol and evaporatedin vacuo to 50% of the original volume. Co-evaporation with n-butanol(3×) was followed by partitioning the crude syrup between ethyl acetateand sat. aqueous NaHCO₃. After back extracting the aq. layer with ethylacetate, the combined organics were evaporated to dryness in vacuo . Thecrude residue was then dissolved in anhydrous dichloromethane (50 ml)and treated with a solution of TEA3HF (27.84 mmol, 4.54 ml) and TEA(8.17 ml), in dichloromethane (20 ml). The reaction mixture wasevaporated to dryness in vacuo and subsequently dissolved in additionaldichloromethane (20 ml). Evaporation followed by dilution was repeated 3times, and the crude product purified by flash chromatography. Agradient of 2-10% ethanol in dichloromethane afforded (14) as lightyellow foam; 3.02 g, 88% yield. ¹H NMR (dmso-d6): 12.08 (s, exch, 1H,NH), 11.63 (s, exch, 1H, NH), 8.29 (s, 1H, H8), 5.90 (d, J_(1′,2′)=6.3,1H, H1′), 5.23 (d, J_(OH,3′)=4.9, 1H, 3′-OH), 5.07 (t, J_(OH,5′)=5.3,J_(OH,5) =5.3, 1H, 5′-OH), 4.30 (m, J_(3′,2′)=4.8, J_(3′,4′)=3.3, 1H,H3′), 4.22 (t, J_(2′,1′)=6.3, J_(2′,3′)=4.8, 1H H2′), 3.93 (m,J_(4′,3′)=3.3, J_(4′,5′)=3.9, J_(4′,5′)=3.9, 1H, H4′) 3.65-3.53 (m, 2H,H5′, H5″), 3.33 (s, 3H, OCH₃), 2.78 (m, 1H, iBu—CH), 1.12 (d, 6H,iBu-(CH₃)₂).

[0107] N²-Isopropylphenoxyacetyl-2′-O-Methylguanosine (15):

[0108] A solution of (12) (47.4 g, 88 mmol) in anhydrous pyridine (500ml) was cooled to −10° C. in an ice/ethanol bath while stirring underargon. Isopropylphenoxyacetyl chloride (96.8 mmol, 20.6 ml) was addeddropwise to the stirred −10° C. solution over a period of 5 minutes. Thereaction mixture was stirred at −10° C. for 2 hours followed by 1 hourat rt then quenched with ethanol (20 ml). After evaporating the reactionmixture to dryness in vacuo, the resulting residue was partitionedbetween dichloromethane and sat. aqueous NaHCO₃. The aqueous layer wasback extracted with dichloromethane and the combined organics dried overNa₂SO₄. Filtration and evaporation of the filtrate in vacuo afforded abeige foam which was dissolved in glacial acetic acid (1000 ml). To thestirred acetic acid solution was added water (400 ml) followed by NaNO₂(742.4 mmol, 51.2 g). Another portion of NaNO₂ (742.4 mmol, 51.2 g) wasadded after 30 minutes and the reaction stirred at rt for 48 hours. Thereaction mixture was diluted with one volume of n-butanol and evaporatedin vacuo to 50% of the original volume. Co-evaporation with n-butanol(3×) was followed by partitioning the crude syrup between ethyl acetateand sat. aqueous NaHCO₃. After back extracting the aq. layer with ethylacetate, the combined organics were evaporated to dryness in vacuo. Thecrude residue was then dissolved in anhydrous dichloromethane (500 ml)and treated with a solution of TEA3HF (278.4 mmol, 45.4 ml) and TEA(81.7 ml), in dichloromethane (200 ml). The reaction mixture wasevaporated to dryness in vacuo and subsequently dissolved in additionaldichloromethane (200 ml). Evaporation followed by dilution was repeated3 times, and the crude product purified by flash chromatography. Agradient of 2-10% ethanol in dichloromethane afforded (5) as lightyellow foam; 29.2 g, 85% yield. ¹H NMR (dmso-d6): 11.65 (s, exch, 2H,NH, NH), 8.30 (s, 1H, H8), 7.18-6.88 (dd, 4H, phenoxy), 5.91 (d,J_(1′,2′)=6.0, 1H, H1′), 5.24 (d, J_(OH,3′)=4.8, 1H, 3′-OH), 5.09 (t,J_(OH,5) =5.6, J_(OH,5″)=5.2, 1H, 5′-OH), 4.82 (s, 2H, CH₂), 4.31 (m,J_(3′,2′)=4.8, J_(3′,4′)=3.6, 1H, H3′), 4.23 (t, J_(2′,1′)=6.0,J_(2′,3′)=4.8, 1H, H2′), 3.93 (m, J_(4′,3′)=3.6, J_(4′,5′)=4.0,J_(4′,5″=)3.9, 1H, H4′) 3.65-3.53 (m, 2H, H5′, H5″), 3.35 (s, 3H, OCH₃),2.84 (m, 1H, iPr—CH), 1.17 (d, 6H, iPr—(CH₃)₂).

[0109] N1-Benzyl Guanosine (16)

[0110] Guanosine hydrate (50 grams, 177 mmol) was coevaporated twicefrom dimethylformamide (2×250 ml) and dissolved in dry dmf (400 mls).N,N-dimethylformamide dibenzyl acetal was added (240 grams, 230 ml, 885mmol) and the solution was heated with stirring to 80° C. for 18 hrs.The excess acetal was removed by steam distillation on a rotaryevaporator. The product was recovered without chromatography by washingwith dichloromethane/hexanes (1:1 v/v) to yield 84 g of the ortho-amideintermediate. The o ortho-amide was cleaved by treatment with aqueoussodium hydroxide (2N, 133 ml) at room temperature for four hours. Theproduct was recrystallized from boiling water to yield 54 g (145 mmol,82%) of pure (16). ¹H NMR (dmso-d₆): 7.97 (s, 1H, H8), 7.29 (m, 5H, Bz),7.02 (bs, 2H, 2NH₂), 5.70 (d, J_(1′,2′)=5.6, 1H, H1′), 5.42 (bs, 1H,2′OH), 5.23 (s, 1H, CH₂-Bz), 5.15 (bs, 1H, 3′OH), 5.00 (bs, 1H, 5′-OH),4.41 (t, J_(3′,2′)=5.6, J_(3′,4′)=4.0, 1H, H3′), 4.08 (t, J_(2′,1′)=6.3,J_(2′,3′)=4.8, 1H, H2′), 3.85 (m, J_(4′,3′)=3.5, J_(4′,5′)=4.0, 1H, H4′)3.58-3.49 (m, 2H, H5′, H5″).

[0111] N1-Benzyl-2′-O-Methyl Guanosine (17)

[0112] A 1 L pear shaped recovery flask with stir bar was charged with amixture of 16 (50 g, 134 mmol), silver acetylacetonate (41 g 200 mmol),TMSH (200 ml of 1N solution in methanol) and dimethylformamide (400 ml).The flask was heated to 70° C. for two hours. The solution was cooled toambient temperature, neutralized to pH 7 with 1 M HCI, and dried to asolid tar. The tar was dissolved in water (500 ml) and filtered througha sintered glass funnel to remove silver salts. The product wasevaporated to dryness to remove water and residual solvents and 10 grams(26 mmols) was redissolved in 50 ml of water prior to purification on aWaters Delta-Pak ODS 50 mm×300 mm HPLC column. The N1-Bz-2′-OMeguanosine isomer eluted first and was recovered using a rotaryevaporator. The product (7 g, 18 mm, 70%) was identical to an authenticsample by HPLC, UV-, ¹H-NMR-spectroscopy. ¹H NMR (dmso-d₆): 8.02 (s,1H,H8), 7.26 (m, 5H, ph), 7.02 (bs, 2H, 2NH2), 5.82 (d, J_(1′,2′)=6.4, 1H,H1′), 5.23 (s, 2H, CH2-Bz), 5.18 (d, J_(OH,3)=4.8, 1H, 3′-OH), 5.04 (t,J_(OH,5′)=5.2, J_(OH,5″)=5.6, 1H, 5′-OH), 4.28 (m, J_(3′,2′)=4.8,J_(3′,4′)=3.2, 1H, H3′), 4.20 (t, J_(2′,1′)=6.4, J_(2′,3′)=4.8, 1H,H2′), 3.90 (m, J_(4′,3′)=3.6, J_(4′,5′)=4.0, 1H, H4′), 3.65-3.53 (m, 2H,H5′, H5″J_(OH,5′)=5.2, J_(5′,5″)=11.6), 3.33 (s, 3H, OCH₃).

[0113] 2′-O-Methyl Guanosine (9)

[0114] Sodium spheres (3.5 grams) in mineral oil were washed withhexanes and weighed into dry THF. A glass bottle with polyethyleneclosure was charged with naphthalene (21.2 g, scintillation grade), dryTHF (210 ml) and a glass sealed stir bar. The sodium was added and themixture was stirred vigorously for one hour. The solution turned darkgreen after ten minutes and all sodium was presumed to be consumed afterone hour to yield 150 mm of 0.6M sodium naphthalene solution. Thissolution was used without further characterization. A 250 ml flask wascharged with N₁Bzl-2′-O-methyl guanosine (2 g, 5.0 mm) and a glasssealed stir bar. Sodium naphthalene solution was added (50 mmol, 90 ml)and the solution was stirred overnight. TLC (10% MeOH in DCM) showedcomplete deblock of the benzyl group. The reaction was quenched with 10mls of methanol and all solvents were removed with a rotary evaporator.Water (100 ml) was added and the solution was neutralized with HCI (1N,pH 7, 50 ml). Naphthalene was removed with extraction by toluene (3×100mls) and the solution was pumped over an ODS Delta-Pak column to recover2′-O-Me guanosine nucleoside. The product (1.3 g, 4.5 mmol, 90%) wasidentical to an authentic sample by HPLC, UV-Vis & ¹H-NMR-spectroscopy.

[0115] Trimethylsulfonium Hydroxide (TMSH) Solution in Methanol:

[0116] A 0.2 M solution of trimethyl sulfonium iodide (TMSI, 102 grams,0.5 mol), in 2.5 L of methanol and water (9:1, v/v) was prepared byheating to 40 C and mixing continuously for 30 min. The clear, colorlesssolution was allowed to cool to r.t. A glass chromatography column (Ace#50 thread; 75 mm×300 mm) containing Duolite 147 anion exchange resin(900 g) in the hydroxide form was previously packed and used for theconversion of TMSI to TMSH. The solution was pumped over the columnusing a gear pump at 100 ml per minute. The resin was washed with anadditional 1 liter of 90% methanol and the total 3.5 liters was reducedin volume to 500 ml using a rotary evaporator with the bath set at 20°C. The solution was checked for the presence of iodide ion usingacidified silver nitrate solution and found to be negative. The solutionwas not characterized further and was stored in a teflon bottle with agas vent at 5° C.

[0117] 2′-O-Methyl Adenosine (8)

[0118] A solution of adenosine (50 g, 187 mmol) in dimethylformamide(400 ml) was prepared by heating to 50° C. with continuous mixing for 10minutes in a 1 L pear shaped recovery flask containing a stir bar.Silver acetylacetonate (58 g, 280 mmol) and TMSH (280 ml of a 1 M)solution was added and the mixture was stirred immediately. The reactionmixture was heated to 75° C. With stirring for 45 minutes. A sample ofthe mixture showed no starting adenosine and two spots of which thepredominant one comigrates with an authentic sample of 2′-OMe adenosine.An HPLC assay showed an nine to one ratio for 2′ to 3′-OMe adenosine Thesolvent was removed by rotary evaporation and 500 ml of water and 250 mlof 1N HCl was added to neutralize the hydroxide (pH 7 by Hydrion strips)prior to filtration on a sintered glass funnel. The water and solventswere removed by rotary evaporation leaving a brown tar. This materialwas purified on a ODS 25 mm×300 mm Delta-Pak HPLC column using water asthe eluant. Overall recovery of 42 grams for a yield of 75% fromadenosine. The product 12 was identical to authentic sample by HPLC,UV-, ¹H-NMR-spectroscopy.

[0119] These examples are meant to be non-limiting and those skilled inthe art will recognize that similar strategies, as described in thepresent invention, can be readily adapted to synthesize other methoxynucleosides and nucleoside analogs and are within the scope of thisinvention.

[0120] Other embodiments are within the following claims. TABLE IMethylation of N¹-Benzyl Guanosine with acethylacetonates

Metal 2′-O—Me-G^(bzl) 3′O—Me-G^(bzl) 2′-O—Me-G yield Cu 40 42 32 Mg 65 7 45 Ag 70  8 70

[0121] TABLE II Metal-Directed methylation of Adenosine

Metal 2′-O—Me-A 3′-O—Me-A 2′-O—Me-A yield Fe 1 1 45% Cu 2 1 60% Ag 4 172% Sr 8 1 80%

1. A process for the synthesis of a 2′-O-methyl adenosine nucleosidephosphoramidite, comprising the steps of a) contacting a solution ofN4-acetyl-5′,3′-di-O-acetyl-2′-O-methyl cytidine with a Lewis acid underconditions suitable for formation of 2′-O-methyl adenosine nucleoside,and b) converting the 2′-O-methyl adenosine nucleoside to a 2′-O-methyladenosine nucleoside phosphoramidite under conditions suitable forformation of said 2′-O-methyl adenosine nucleoside phosphoramidite.
 2. Aprocess for the synthesis of 2′-O-methyl guanosine nucleosidephosphoramidite, comprising the steps of: a) methylating2-amino-6-chloropurine riboside by contacting said 2amino-6-chloropurineriboside with sodium hydride, dimethylformamide and methyl iodide underconditions suitable for formation of 2′-O-methyl-2-amino-6-chloropurineriboside; b) contacting said 2′-O-methyl-2-amino-6-chloropurine ribosidewith 1,4-diazabicyclo(2.2.2) octane and water under conditions suitablefor formation of said 2′-O-methyl guanosine nucleoside in a crude form;c) purifying said 2′-O-methyl guanosine nucleoside from said crude form;and d) converting said 2′-O-methyl guanosine nucleoside to a 2′-O-methylguanosine nucleoside phosphoramidite under conditions suitable forformation of said 2′-O-methyl guanosine nucleoside phosphoramidite.
 3. Aprocess for the synthesis of 2′-O-methyl adenosine nucleosidephosphoramidite, comprising the steps of: a) methylating2-amino-6-chloropurine riboside by contacting said2-amino-6-chloropurine riboside with sodium hydride, dimethylformamideand methyl iodide under conditions suitable for formation of2′-O-methyl-2-amino-6-chloropurine riboside; b) contacting said2′-O-methyl-2-amino-6-chloropurine riboside with acetic anhydride,4-dimethylaminopyridine and triethylamine under conditions suitable forformation of 3′,5′-di-O-acetyl-2′-O-methyl-6-chloro-2-aminopurineriboside; c) deaminating said3′,5′-di-O-acetyl-2′-O-methyl-6-chloro-2-aminopurine riboside withisoamyl nitrite and tetrahydrofuran to form3′,5′-di-O-acetyl-2′-O-methyl-6′-chloropurine; d) aminating said3′,5′-di-O-acetyl-2′-O-methyl-6-chloropurine with ammonia to form2′-O-methyl adenosine nucleoside in a crude form; e) purifying said2′-O-methyl adenosine nucleoside from said crude form; and f) convertingsaid 2′-O-methyl adenosine nucleoside to a 2′-O-methyl adenosinenucleoside phosphoramidite under conditions suitable for formation ofsaid 2′-O-methyl adenosine nucleoside phosphoramidite.
 4. A process forthe synthesis of N2-isobutyryl-2′-O-methyl guanosine nucleosidephosphoramidite, comprising the steps of: a) contacting2,6-diaminopurine nucleoside with anhydrous pyridine and1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane under conditions suitablefor formation of2,6-diamino-9-(3,5-O-tetraisopropyidisiloxan-(1,3-diyl)-beta-D-ribofuranosyl)purine; b) methylating said2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-beta-D-ribofuranosyl)purine by contacting said 2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-beta-D-ribofuranosyl) purine withanhydrous DMF and methyl iodide under conditions suitable for formationof2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2′-O-methyl-beta-D-ribofuranosyl)purine; c) acylating said2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2-O-methyl-beta-D-ribofuranosyl)purine by contacting said 2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2-O-methyl-beta-D-ribofuranosyl)purine with anhydrous pyridine and isobutyryl chloride under conditionssuitable for formation of2,6-diamino-N2-isobutyryl-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2-O-methyl-beta-D-ribofuranosyl) purine; d) deaminating and desilylatingsaid 2,6-Diamino-N2-isobutyryl-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2-O-methyl-beta-D-ribofuranosyl)purine under conditions suitable for formation ofN2-isobutyryl-2′-O-methyl guanosine nucleoside in a crude form; e)purifying said N2-isobutyryl-2′-O-methyl guanosine nucleoside from saidcrude form; and f) converting said N2-isobutyryl-2′-O-methyl guanosinenucleoside to a N2-isobutyryl-2′-O-methyl guanosine nucleosidephosphoramidite under conditions suitable for formation of saidN2-isobutyryl-2′-O-methyl guanosine nucleoside phosphoramidite.
 5. Aprocess for the synthesis of N2-isopropylphenoxyacetyl-2′-O-methylguanosine nucleoside phosphoramidite, comprising the steps of: a)contacting 2,6-diaminopurine nucleoside with anhydrous pyridine and1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPSCI) under conditionssuitable for formation of 2,6-diamino-9-(3′,5′-O-tetraisopropyldisiloxan-(1,3-diyl) -beta-D-ribofuranosyl) purine;b) methylating said2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-beta-D-ribofuranosyl)purine by contacting said 2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-beta-D-ribofuranosyl) purine withanhydrous DMF and methyl iodide under conditions suitable for formationof2,6-diamino-9-(3′,5′-O-tetraisopropyldisiloxan-(1,3-diyl)-2′-O-methyl-beta-D-ribofuranosyl)purine; c) acylating said2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2-O-methyl-beta-D-ribofuranosyl)purine by contacting said 2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2-O-methyl-beta-D-ribofuranosyl)purine with anhydrous pyridine and isopropylphenoxyacetyl chloride underconditions suitable for formation of2,6-diamino-N2-isopropylphenoxyacetyl-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2-O-methyl-beta-D-ribofuranosyl)purine; d) deaminating and desilylating said2,6-diamino-N2-isopropylphenoxyacetyl-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2-O-methyl-beta-D-ribofuranosyl)purine under conditions suitable for formation ofN2-isopropylphenoxyacetyl-2′-O-methyl guanosine nucleoside in a crudeform; e) purifying said N2-isopropylphenoxyacetyl-2′-O-methyl guanosinenucleoside from said crude form; and f) converting saidN2-isopropylphenoxyacetyl-2′-O-methyl guanosine nucleoside to aN2-isopropylphenoxyacetyl-2′-O-methyl guanosine nucleosidephosphoramidite under conditions suitable for formation of saidN2-isopropylphenoxyacetyl-2′-O-methyl guanosine nucleosidephosphoramidite.
 6. A process for the synthesis of 2′-O-methyl guanosinenucleoside phosphoramidite, comprising the steps of: a) contactingguanosine with N,N-dimethylformamide dibenzyl acetal under conditionssuitable for formation of N1-benzyl guanosine; b) methylating saidN1-benzyl guanosine by contacting said N1-benzyl guanosine with silveracetylacetonate, trimethylsulphonium hydroxide and dimethylformamideunder conditions suitable for formation of N1-benzyl-2′-O-methylguanosine in a crude form; c) purifying said N1-benzyl-2′-O-methylguanosine from said crude form; d) removing the N1-benzyl protectionfrom said N1-benzyl-2′-O-methyl guanosine by contacting saidN1-benzyl-2′-O-methyl guanosine with sodium naphthalene under conditionssuitable for formation of 2′-O-methyl guanosine nucleoside in a crudeform; e) purifying said 2′-O-methyl guanosine nucleoside from said crudeform; and f) converting said 2′-O-methyl guanosine nucleoside to a2′-O-methyl guanosine nucleoside phosphoramidite under conditionssuitable for formation of said 2′-O-methyl guanosine nucleosidephosphoramidite.
 7. A process for the synthesis of 2′-O-methyl adenosinenucleoside phosphoramidite, comprising the steps of: a) methylatingadenosine by contacting said adenosine with dimethylformamide, silveracetylacetonate and trimethylsulphonium hydroxide under conditionssuitable for formation of 2′-O-methyl adenosine nucleoside in a crudeform; b) purifying said 2′-O-methyl adenosine nucleoside from said crudeform; and c) converting said 2′-O-methyl adenosine nucleoside to a2′-O-methyl adenosine nucleoside phosphoramidite under conditionssuitable for formation of said 2′-O-methyl adenosine nucleosidephosphoramidite.
 8. A process for the synthesis of 2′-O-methyl guanosinenucleoside phosphoramidite, comprising the steps of: a) contactingguanosine with N,N-dimethylformamide dibenzyl acetal under conditionssuitable for formation of N1-benzyl guanosine; b) methylating saidN1-benzyl guanosine by contacting said N1-benzyl guanosine withmagnesium acetylacetonate, trimethylsulphonium hydroxide anddimethylformamide under conditions suitable for formation ofN1-benzyl-2′-O-methyl guanosine in a crude form; c) purifying saidN1-benzyl-2′-O-methyl guanosine from said crude form; d) removing theN1-benzyl protection from said N1-benzyl-2′-O-methyl guanosine bycontacting said N1-benzyl-2′-O-methyl guanosine with sodium naphthaleneunder conditions suitable for formation of 2′-O-methyl guanosinenucleoside in a crude form; e) purifying said 2′-O-methyl guanosinenucleoside from said crude form; and f) converting said 2′-O-methylguanosine nucleoside to a 2′-O-methyl guanosine nucleosidephosphoramidite under conditions suitable for formation of said2′-O-methyl guanosine nucleoside phosphoramidite.
 9. A process for thesynthesis of 2′-O-methyl adenosine nucleoside phosphoramidite,comprising the steps of: a) methylating adenosine by contacting saidadenosine with dimethylformamide, magnesium acetylacetonate andtrimethylsulphonium hydroxide under conditions suitable for formation of2′-O-methyl adenosine nucleoside in a crude form; b) purifying said2′-O-methyl adenosine nucleoside from said crude form; and c) convertingsaid 2′-O-methyl adenosine nucleoside to a 2′-O-methyl adenosinenucleoside phosphoramidite under conditions suitable for formation ofsaid 2′-O-methyl adenosine nucleoside phosphoramidite.
 10. A process forthe synthesis of 2′-O-methyl adenosine nucleoside phosphoramidite,comprising the steps of: a) methylating adenosine by contacting saidadenosine with dimethylformamide, strontium acetylacetonate andtrimethylsulphonium hydroxide under conditions suitable for formation of2′-O-methyl adenosine nucleoside in a crude form; b) purifying said2′-O-methyl adenosine nucleoside from said crude form; and c) convertingsaid 2′-O-methyl adenosine nucleoside to a 2′-O-methyl adenosinenucleoside phosphoramidite under conditions suitable for formation ofsaid 2′-O-methyl adenosine nucleoside phosphoramidite.
 11. A process forthe synthesis of N2-isobutyryl-2′-O-methyl guanosine nucleosidephosphoramidite, comprising the steps of: a) contacting2,6-diaminopurine nucleoside with anhydrous pyridine and TIPSCI underconditions suitable for formation of 2,6-diamino-9-(3′,5′-O-tetraisopropyldisiloxan-(1,3-diyl)-.beta.-D-ribofuranosyl) purine; b)methylating said2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-beta-D-ribofuranosyl)purine by contacting said 2,6-diamino-9-(3′,5′-O-tetraisopropyldisiloxan-(1,3-diyl)-beta-D-ribofuranosyl) purine withanhydrous DMF and methyl iodide under conditions suitable for formationof2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2-O-methyl-beta-D-ribofuranosyl)purine; c) acylating said2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2-O-methyl-beta-D-ribofuranosyl)purine by contacting said 2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2-O-methyl-beta-D-ribofuranosyl)purine with anhydrous pyridine and isobutyryl chloride under conditionssuitable for formation of2,6-diamino-N2-isobutyryl-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2-O-methyl-beta-D-ribofuranosyl) purine; d) deaminating and desilylatingsaid 2,6-diamino-N2-isobutyryl-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2′-O-methyl-beta-D-ribofuranosyl)purine under conditions suitable for formation ofN2-isobutyryl-2′-O-methyl guanosine nucleoside in a crude form; e)purifying said N2-isobutyryl-2′-O-methyl guanosine nucleoside from saidcrude form; and f) converting said N2-isobutyryl-2′-O-methyl guanosinenucleoside to a N2-isobutyryl-2′-O-methyl guanosine nucleosidephosphoramidite under conditions suitable for formation of saidN2-isobutyryl-2′-O-methyl guanosine nucleoside phosphoramidite.
 12. Aprocess for the synthesis of N2-isopropylphenoxyacetyl-2′-O-methylguanosine nucleoside phosphoramidite, comprising the steps of: a)contacting 2,6-diaminopurine nucleoside with anhydrous pyridine andTIPSCI under conditions suitable for formation of 2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-beta-D-ribofuranosyl) purine; b)methylating said2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-beta-D-ribofuranosyl)purine by contacting said 2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-beta-D-ribofuranosyl) purine withanhydrous DMF and methyl iodide under conditions suitable for formationof2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2-O-methyl-beta-D-ribofuranosyl)purine; c) acylating said2,6-diamino-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2-O-methyl-beta-D-ribofuranosyl)purine by contacting said 2,6-diamino9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2′-O-methyl-beta-D-ribofuranosyl)purine with anhydrous pyridine and isopropylphenoxyacetyl chloride underconditions suitable for formation of2,6-Diamino-N2-isopropylphenoxyacetyl-9-(3′,5′-O-tetraisopropyldisiloxan-(1,3-diyl)-2′-O-methyl-beta-D-ribofuranosyl)purine; d) deaminating and desilylating said2,6-diamino-N2-isopropylphenoxyacetyl-9-(3,5-O-tetraisopropyldisiloxan-(1,3-diyl)-2-O-methyl-beta-D-ribofuranosyl)purine under conditions suitable for formation ofN2-isopropylphenoxyacetyl-2′-O-methyl guanosine nucleoside in a crudeform; e) purifying said N2-isopropylphenoxyacetyl-2′-O-methyl guanosinenucleoside from said crude form; and f) converting saidN2-isopropylphenoxyacetyl-2′-O-methyl guanosine nucleoside to aN2-isopropylphenoxyacetyl-2′-O-methyl guanosine nucleosidephosphoramidite under conditions suitable for formation of saidN2-isopropylphenoxyacetyl-2′-O-methyl guanosine nucleosidephosphoramidite.